This invention relates to a method for the in vitro testing of chemicals to determine reproductive toxicity, and a method for producing, in vitro, rabbit spermatozoa of hyperactivated motility useful in said testing.
The present invention provides a method for in vitro toxicity testing through exposure of hyperactivated spermatozoa to test chemicals and measuring the inhibition of hyperactivated motility. This invention also provides a process for producing spermatozoa of hyperactivated motility in one-half to one hour, thereby making it practical to perform said testing.
Development of alternative toxicological testing methods which do not require harm to animals is highly desirable in view of the current societal attitude towards the use of animals in experiments. Methods currently used to detect male reproductive toxicants are wanting in this respect in that they require the exposure of chemicals to large numbers of animals. Moreover, the methods used are time consuming and costly.
Spermatozoa are capable of fertilizing an egg only after undergoing a process in the female reproductive tract known as capacitation. An integral part of capacitation is the development by spermatozoa of hyperactivated motility, characterized by a low frequency, wide amplitude bending of the tail resulting in vigorous random and non-progressive movements. Development of hyperactivated motility is generally recognized as a prerequisite to fertilization. The inhibition of hyperactivity by xenobiotics indicates the xenobiotic will adversely affect fertility.
Any chemical that adversely affects spermatozoan motility is suspect as a reproductive toxicant which may prevent fertilization. Measurement of the extent of hyperactivated motility development in spermatozoa in the presence of chemicals provides a means for the in vitro assessment of the chemical's potential to cause male fertility disturbances.
The invention described herein employs a simple salts medium and incubation protocol which induces hyperactivated motility in spermatozoa in one-half to one hour. Motility parameters are then measured using existing motion analysis systems such as CellSoft or CellTrak, and using previously formulated models for classification of spermatozoa, the extent of hyperactivation development in the presence or absence of the test chemical is computed. The ability to objectively recognize hyperactivated spermatozoa enables the measurement of hyperactivated motility inhibition by chemicals. This in vitro method is faster and more economical than traditional animal testing methods and does not harm or expose animals to chemicals.